Cells in our body have certain preferential orientation for proper physiological functions. Vascular smooth muscle cells are oriented so that blood vessels are functionally adaptable (i.e., capable of tone control). Understanding basic molecular mechanism that regulate orientation of smooth muscle will lead to better technologies for vascular tissue engineering. Specifically, this study will focus on roles of fibronectin-mediated cell adhesion in orientation of smooth muscle cells. We have developed a unique in vitro model system in which endothelial cells under mechanical loading are used to synthesize endogenous oriented ECM fibers. Preliminary study demonstrated that smooth muscle orientation was guided by the oriented endothelial ECM. We also developed tools that quantitate the degree of contact guidance on cell orientation. This model system and tools are ready to accomplish following specific aims. Specific Aim 1: To determine which cell binding sequence in fibronectin is necessary to guide orientation of smooth muscle cells. Three sites in the cell binding domain (motifs containing RGD, PHSRN, and LDV sequences) cooperatively mediate fibronectin-dependent cell adhesion. Using inhibitory antibodies and synthetic peptides, each motif in the cell binding domain will be independently blocked. We will quantitate effects of the blocking on orientation of smooth muscle cells cultured on the conditioned ECM. Combinatory effects of the blocking will also be determined. Specific Aim 2: To determine specificity of fibronectin receptors for determination of smooth muscle orientation. We hypothesize that specific integrin receptors are necessary to guide orientation of smooth muscle cells on the conditioned ECM. Two fibronectin receptors (integrin a4b1 and a5b1) will be tested using three complementary methods as follows: 1) block fibronectin receptors by inhibitory antibody, 2) activate fibronectin receptors by stimulatory antibodies, and 3) overexpress fibronectin receptors by gene transfection.